Anti-Cancer Activity of Catechin against A549 Lung Carcinoma Cells by Induction of Cyclin Kinase Inhibitor p21 and Suppression of Cyclin E1 and P–AKT

Round 1

Reviewer 1 Report

This paper describes influence of cathechin on proliferation of A549 cancer cells and its influence on the production of cyclin-dependent kinase inhibitors p21 and p27. The problem with this paper is that antiproliferative activity is rather low and maybe in connection with elevated level of the two p21 and p27 proteins there could be some beneficiary effect. Paper would be easier to read if Authors could provide scheme of interations they are reporting on and to equipp paper with list of abbreviations.

Data required in lines 206-238 should be given.

Author Response

The authors thanked the reviewer pointing out the importance of this study.  The replies to the reviewer’s comment was listed below:

1. The problem with this paper is that antiproliferative activity is rather low and maybe in connection with elevated level of the two p21 and p27 proteins there could be some beneficiary effect. Paper would be easier to read if Authors could provide scheme of interactions they are reporting on and to equipp paper with list of abbreviations.

Reply: The p21 and p27 proteins are responsible for regulating cell cycle, however, other upregulation factors might also be involved to compensate A549 cell proliferation inhibition. Thus, in the future study, we are going to investigate other regulation factor and pathway to explore if there is crosstalk between different pathways affecting the cancer cell proliferation.

                  The list of abbreviation has been attached as supplementary material

2. Data required in lines 206-238 should be given.

        Reply: The information has been added in the manuscript and highlighted.

Reviewer 2 Report

The paper of H. Sun et.al. study the biological effect of catechin on the A549 lung cancer cell lines. The particular attention is pointed towards the mechanism of action of the catechin in this cell line.

Catechin shows some inhibitory effect of the A549 cells grow at 600 microM concentration (that is definitely too high). Unfortunately, there are no any data presented of catechin effect on non-transformed cells (e.g. BEAS-2B cell line). The lack of such a comparison makes the statement about the antitumor activity completely unfounded. These experiments have to be done before publishing.

The protein expression after the treatment with catechin was poorly affected in case of p21 and unaffected in case of p27. Authors have to add the standard error bar to the control bar. Again, the same experiment have to be repeated with non-transformed cells. As there are no correlation of catechin treatment and p27 expression authors have to change the title of the manuscript.

In case of Cycline E1 and P-AKT expression experiment, at the first glance the western blot assay does not correspond to the expression level graphs. In particular, the CyclineE and AKT expression level seems to be higher in the cells treated with 600 microM catechin than in the control experiment (with almost the same level of actin expression). Probably the quality of western blot image should be improved. Again, the same experiment should be repeated with non transformed cells.

This paper is too immature and should be improved before considering for publishing.

Author Response

The authors thanked the reviewer pointing out the importance of this study.  The replies to the reviewer’s comment was listed below:

1. Catechin shows some inhibitory effect of the A549 cells grow at 600 microM concentration (that is definitely too high). Unfortunately, there are no any data presented of catechin effect on non-transformed cells (e.g. BEAS-2B cell line). The lack of such a comparison makes the statement about the antitumor activity completely unfounded. These experiments have to be done before publishing.

Reply: Catechin is a common bioactive compound in various food, especially in tea.  Since catechin concentration in tea is comparable or even higher than our studied concentration, it is considered to be safe.  Thus, we did not use normal cell as comparison. 

2. The protein expression after the treatment with catechin was poorly affected in case of p21 and unaffected in case of p27. Authors have to add the standard error bar to the control bar. Again, the same experiment have to be repeated with non-transformed cells. As there are no correlation of catechin treatment and p27 expression authors have to change the title of the manuscript.

Reply: The error bar has been added to the control group in Fig.2. 

The title has been modified.

3. In case of Cycline E1 and P-AKT expression experiment, at the first glance the western blot assay does not correspond to the expression level graphs. In particular, the CyclineE and AKT expression level seems to be higher in the cells treated with 600 microM catechin than in the control experiment (with almost the same level of actin expression). Probably the quality of western blot image should be improved. Again, the same experiment should be repeated with non transformed cells.This paper is too immature and should be improved before considering for publishing.Reply:The images of Cycline E1 and P-AKT have been replaced by their replicate sample images and the resolution should be improved.

Reviewer 3 Report

Sun et al. reported the anti-cancer characteristic of catechin, in which catechin suppresses the proliferation of A549 non-small lung cancer cells in a dose- and time-dependent manner. This anti-cancer activity was attributed to the capability of catechin in increasing the expression of cyclin kinase inhibitor p21 and p27 while suppressing the expression of cyclin E1 and P-AKT. Overall, this study is quite interesting as it studied and proposed the mechanisms driving the anti-cancer activity of catechin. However, more work should be done to improve its quality to be reconsidered for publication in Applied Sciences.

 

1. For Figure 1b, what was the concentration of catechin? The authors should clarify this in the main text and figure caption.
2. In Table 2, the * indicates significant difference between two data for p < 0.05. However, it is unclear which two data sets were being compared. The authors should clarify this.
3. The authors claimed that catechin also increased the expression of p27, but the difference in the relative expression levels of p27 for control and treatment groups was not statistically significant. The authors should provide a clearer explanation on this.

Author Response

1. The concentration of catechin in Fig 1b has been added and highlighted in green.
2. The * indicated significant difference between treatment and control. It has been clarified in the text and highlighted.
3. The author stated “No significant incensement of p27 expression was observed in catechin treatments and control group.” in line 162.

Round 2

Reviewer 1 Report

Now, after explanation and corrections paper could be published

Author Response

The authors all thanked the reviewer's comment.

Reviewer 2 Report

The main drawback of the manuscript persists: there is no comparison of the toxicity of catechin in the cancer and non-transformed cells. In their reply authors state “catechin is a common bioactive compound in various food, especially in tea. Since catechin concentration in tea is comparable or even higher than our studied concentration, it is considered to be safe”.

I must disagree with such a statement: the concentration of catechin in food and its concertation in cellular medium could not be compared. At such a high concentration catechin could have cytotoxic effect in non-transformed cells (see e.g. Silvia C. et.al., European Journal of Pharmacology 859 (2019) 172533).

For my opinion, the paper could not be published without the additional tests on non-transformed cells.

Author Response

The purpose of this study is to investigate the target site and trend for the anticancer capability of catechin.  Thus, in our future study, we will narrow down the range of the concentration to find the best anticancer potency and use non-transformed cells as comparison.